Immunogenic fragment peptide of EN2 protein or antibody composition specifically recognizing same

ABSTRACT

The present invention relates to an immunogenic fragment peptide of an EN2 protein or an antibody composition specifically recognizing the same. In the present invention, EN2 protein can be quantified through a method of specifically recognizing the peptide. Also, an antibody prepared using the peptide has vastly superior detection sensitivity compared to existing EN2 protein antibodies and thus can be useful in a diagnostic agent for diagnosing prostate cancer.

TECHNICAL FIELD

The present invention relates to an immunogenic fragment peptide of anEN2 protein or an antibody composition specifically recognizing thesame.

BACKGROUND ART

The prostate is a walnut-sized male reproductive organ beneath thebladder and in front of the rectum, and functions to create semen andstore a portion thereof. The upper part of the prostate is adjacent tothe bladder neck that connects from the bladder to the urethra and isfixed to the anterior puboprostatic ligament, and the lower part thereofis fixed by the genitourinary diaphragm. Most cancer that occurs in theprostate is adenocarcinoma (gland cell carcinoma) that forms in prostatecells. The types of cancer may be classified depending on the degree ofdifferentiation of the tumor tissue and the characteristics of thecells.

Prostate cancer is one of the most common genitourinary tumors in theworld. In the United States, about 180,890 persons were newly diagnosedwith prostate cancer in 2016, accounting for 10.7% of all new tumordiagnoses in the United States, and prostate cancer, following breastcancer and lung cancer, is the third most frequently occurring kind oftumor. Based on worldwide statistics from 2009 to 2013, 129.4 men among100,000 men have prostate cancer, and, based on statistics from 2016,26,120 prostate cancer patients died. Moreover, prostate cancer is anuncommon disease before age 50, but shows a rapid increase after age 50.Recently, the number of aged men is drastically increasing in Korea dueto the prolongation of the life expectancy and thus continuousmanagement is required in order to prevent aggravation of the prostatecancer, such as metastasis, etc., through early diagnosis.

In particular, human prostate cancer appears to have a tendency tometastasize to the bone, and is known to inevitably progress from anandrogen-dependent state to an androgen-resistant state, therebyincreasing a patient mortality rate. Furthermore, about 25% of men whohave undergone prostate cancer therapy require additional treatment dueto recurrence of the disease, and prostate cancer is currently thesecond leading cause of cancer-related death in men in the UnitedStates. Hence, early diagnosis and treatment of prostate cancer isnecessary.

Examples of a direct prostate cancer diagnosis method currently in useinclude a method of directly imaging the prostate or a biopsy diagnosismethod. In the case of diagnosis through direct imaging or biopsy, it isdifficult to diagnose the onset of prostate cancer at the initial stage,and thus it is urgently necessary to develop a method for in-vitrodiagnosis.

As an indirect method, there is a diagnosis method that may be performedin vitro using a prostate-specific antigen (PSA) assay. However, the PSAused for diagnosis is produced not only in malignant prostate epitheliumbut also in normal and benign tissues, resulting in a highfalse-positive rate in prostate cancer detection. In addition, asignificant increase in serum PSA level may be used for an effectivestandard method for diagnosing prostate cancer, whereas a weak increasein PSA serum level on the order of 2 to 10 ng/mL makes it impossible toreach a definite prostate cancer diagnosis. In the case of such a weakincrease, the serum PSA may originate from a non-tumorous disease suchas benign prostatic hyperplasia (BPH), prostatitis or other physicaltrauma, and PSA analysis for prostate cancer diagnosis has problemsrelated to detection specificity.

Accordingly, diagnosis of prostate cancer using a new biomarker isregarded as important, and research thereon has been conducted (KoreanPatent Application Publication No. 10-2009-0111307) but is stillinsufficient. Recently, methods of diagnosing prostate cancer using EN2(engrailed-2) as a biomarker have been proposed, and EN2 is well knownas a biomarker that is capable of solving the detection specificityproblem of PSA (prostate specific antigen) assay used in conventionalprostate cancer diagnosis. EN2 protein acts as a transcription factor incells and is overexpressed only in prostate cancer cells, undesirablycausing a DNA transcriptional regulatory disorder. Furthermore, when EN2expression increases in prostate cancer cells, the amount of EN2 proteinthat is excreted by the urine is also increased, whereby EN2 is suitablefor use in in-vitro analysis. Accordingly, U.S. Pat. No. 8,460,882,Japanese Patent Application Publication No. 2012-532621 and U.S. Pat.No. 8,722,643 disclose a diagnostic composition recognizing an EN2protein, and Korean Patent Application Publication No. 10-2016-0077788discloses a DNA aptamer specifically binding to EN2.

Generally, the term “antibody” means that an external substance (asubstance having at least a predetermined size and conditions) (anantigen) is injected into an organism, and an antibody having a site(epitope) capable of specifically recognizing the external substancethrough a humoral immune response is formed in the organism, and is thencollected from the blood and separated from the serum. The antibody thusformed is a polyclonal antibody having multiple sites capable ofrecognizing the antigen.

In the humoral immune response, antigens are presented to B cellsthrough epitopes by various antigen-presenting cells (APC) and activateB cells. During the activation process, B cells are differentiated intoplasma cells that enable only a single antibody to be produced in alarge amount and secreted ex vivo by rearranging only the genes thatmake the antibody that is able to react with the antigen, among thewhole genes that make the antibody, and removing the remainingunnecessary genes.

Since antigens typically have multiple epitopes, there are various kindsof differentiated plasma cells, and thus various antibodies areproduced. The entire set of diverse antibodies secreted from variouskinds of plasma cells (i.e. genes that make the antibody are different)is called a “polyclonal antibody”, and only a single antibody made fromone kind of plasma cell is called a “monoclonal antibody”.

The market for diagnostic products using antibodies has been rapidlyincreasing since 1980, and antibodies are highly sensitive enough todetect very small amounts of proteins specifically expressed dependingon diseases or symptoms, and thus are utilized for the development ofhighly efficient diagnostic kits and diagnosis methods. To this end, itis essential to develop an antibody that has both specificity andsensitivity to a protein (antigen) expressed depending on diseases orsymptoms. In order to produce antibodies having specificity andsensitivity, the efficiency of the antibody may be maximized byconsidering several factors.

First, the primary sequence of an antigen to be produced is compared,and thus an animal to be immunized, having high heterogeneity fromanimal species of the antigen, is selected, thereby maximizing theimmune response. For example, when the sequence of a human protein isused, an animal having high heterogeneity from the above sequence may beselected for induction of an immune response, thereby obtaining ahigh-titer antibody.

Second, selection is performed taking into consideration the fact thatthe quality of the antigen may vary depending on the characteristics ofthe modification after translation of the antigen sequence and on thestereostructure. In particular, selection has to be conductedconsidering the case where not the whole protein but some peptide formthereof is used as an antigen.

Third, the antibody is prepared so as to be adapted for the purposethereof. A polyclonal antibody is advantageous because it is easy toproduce, facilitates the detection of an antigen due to the wide varietyof epitopes thereof, and enables selection of animals to be immunizedfrom among a wide range depending on the antigen, but is low in antibodyspecificity and is unsuitable for mass production of a consistent titer.Also, a monoclonal antibody has the advantage of producing antibodies inculture supernatants to thus enable mass production of antibodies havingspecificity of a consistent titer but is disadvantageous in that ittakes a long period of production, is limited with respect to the rangeof animals capable of being immunized, and is not suitable forexperiments such as immunological staining.

Therefore, in general, the specificity of the antigen is confirmedthrough the production of a polyclonal antibody, and a monoclonalantibody is prepared when mass production of an antibody having aconsistent titer is required.

Meanwhile, the present inventors have studied compositions related tothe diagnosis of prostate cancer and have prepared a composition fordiagnosing prostate cancer based on an EN2 protein fragment capable ofmore effectively diagnosing an EN2 protein or an antibody capable ofrecognizing the fragment, thus culminating in the present invention.

CITATION LIST

-   (Patent Document 1) U.S. Pat. No. 8,460,882 (Title: Cancer    biomarkers, Applicant: The University of Surrey, Registration date:    Jun. 11, 2013)-   (Patent Document 2) U.S. Pat. No. 8,722,643 (Title: Targeting EN2,    PAX2, and/or DEFB1 for treatment of prostate conditions, Applicant:    Phigenix, Inc., Registration date: May 13, 2014)-   (Patent Document 3) Japanese Patent Application Publication No.    2012-532621 (Title: Therapeutic peptide, polypeptide and nucleic    acid array, Applicant: The University of Surrey, Laid-open date:    Dec. 20, 2012)-   (Patent Document 4) Korean Patent Application Publication No.    10-2009-0111307 (Title: DNA aptamer specifically binding to EN2 and    use thereof, Applicant: POSTECH Research and Business Development    Foundation, Laid-open date: Jul. 4, 2016)-   (Patent Document 5) Korean Patent Application Publication No.    10-2016-0077788 (Title: Prostate-specific transcripts and their use    in the treatment and diagnosis of prostate cancer, Applicant: Exxon    Heat Therapeutics SA, Laid-open date: Oct. 26, 2009)

DISCLOSURE Technical Problem

Accordingly, the present invention is intended to provide an immunogenicfragment peptide of an EN2 protein or an antibody compositionspecifically recognizing the same.

Technical Solution

The present invention provides a peptide comprising the amino acidsequence of SEQ ID NO: 1, 2 or 3 below.

SEQ ID NO: 1:  PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS SEQ ID NO: 2: CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR SEQ ID NO: 3: PRSRKPKKKNPNKEDKRPRTAFTAEQLQR

The peptide may be an immunogenic fragment of EN2 (engrailed-2) protein(Accession No. NP 001418.2).

Thus, the present invention provides a method of diagnosing the presenceor absence of EN2 (engrailed-2) protein by specifically recognizing theabove peptide.

In addition, the present invention provides an antibody compositionspecifically recognizing each peptide comprising the amino acid sequenceof SEQ ID NO: 1, 2 or 3.

Thus, the present invention provides a diagnostic agent for diagnosingprostate cancer containing the above antibody composition.

Thus, the present invention provides a method of diagnosing prostatecancer using the above antibody.

Hereinafter, a detailed description will be given of the presentinvention.

The present invention pertains to a peptide comprising the amino acidsequence of SEQ ID NO: 1, 2 or 3 below.

SEQ ID NO: 1:  PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS SEQ ID NO: 2: CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR SEQ ID NO: 3: PRSRKPKKKNPNKEDKRPRTAFTAEQLQR

In the present invention, commercially available W antibodies, servingas comparative examples, are those prepared using, as an antigen, apeptide comprising the amino acid sequence of SEQ ID NO: 4 or 5 below.

SEQ ID NO: 4:  EDKRPRTAFTAEQLQRLKAEFQTNRYLTE SEQ ID NO: 5:GTCCAGAGGGRGGGAGGEGGASGAEGGGGAGGSEQLLGSGSREPRQNPPCAPGAGGPLPAAGSDSPGDGEGGSKTLSLHGGAKKGGDPGGPLDGSLKARGLGGGDLSVSSDSDSSQAGANLGAQP

The positions of these peptides in EN2 are shown in FIG. 1.

The peptide comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3is an immunogenic fragment of an EN2 protein and is an EN2 proteinfragment having at least one epitope that may be recognized by anantibody for an EN2 protein, a quantity of which increases or decreasesin the body of a prostate cancer patient.

The present invention pertains to a method of diagnosing the presence orabsence of an EN2 protein by specifically recognizing the above peptide.Here, the EN2 protein may be any example thereof extracted from theinside of a mammal to the outside, and may also be a recombinant EN2protein. In order to evaluate the presence or absence of an EN2 protein,any method may be used, so long as it is an ordinary experimental methodused to determine the presence or absence of a protein or in thequantification process thereof.

In addition, the present invention pertains to an antibody compositionspecifically recognizing each peptide comprising the amino acid sequenceof SEQ ID NO: 1, 2 or 3 or to a diagnostic agent containing the same.The diagnostic agent may also contain a labeled secondary antibody, achromophore, an antibody-conjugated enzyme and an additional substanceable to bind to the substrate or antibody thereof.

When the diagnostic agent contains all of respective antibodies forthree types of peptides comprising the amino acid sequences of SEQ IDNOS: 1, 2 and 3, prostate cancer may be more accurately diagnosedcompared to when using a diagnostic agent containing any one or twoantibodies. For example, in order to identify the EN2 protein containedin the body of a subject to be diagnosed, samples collected from thesubject to be diagnosed may be divided into three sets. For one set, thepresence and concentration of EN2 protein are determined using anantibody against the peptide comprising the amino acid sequence of SEQID NO: 1, and for each of the remaining sets, the presence andconcentration of EN2 protein are determined separately for respectiveantibodies against the peptide comprising the amino acid sequence of SEQID NO: 2 and against the peptide comprising the amino acid sequence ofSEQ ID NO: 3, and a total of three EN2 protein detection experiments maybe performed. In this way, when the EN2 protein is detected using thediagnostic agent, the accuracy and reliability of the diagnosis resultmay be increased.

Also, the diagnostic agent of the present invention may contain arecombinant EN2 protein able to quantify the EN2 protein contained inthe body of the subject to be diagnosed.

An antibody is a specific protein molecule directed to an antigenicsite. Therefore, the antibody is preferably an antibody thatspecifically binds to a peptide of the present invention, and mayinclude all of a polyclonal antibody, a monoclonal antibody, and arecombinant antibody.

The antibody may be easily produced using techniques well known in theart. Accordingly, the present invention pertains to a method ofpreparing an antibody using the peptide of the present invention.

The polyclonal antibody may be obtained from the serum obtained byinjecting an animal with the peptide of the present invention as anantigen. The animal may be any animal host such as goat, rabbit, pig andthe like. The monoclonal antibody may be prepared using a hybridomaprocess (Kohler G. and Milstein C.) or a phage antibody library process(Clackson et al. Marks et al.), as widely known in the art to which thepresent invention belongs. The hybridoma process may be conducted usingcells of an immunologically relevant host animal, such as a mouse, and acancer or myeloma cell line. Then, through a process using polyethyleneglycol, as widely known in the art to which the present inventionbelongs, the two kinds of cells are fused, after which theantibody-producing cells may be proliferated through a standard tissueculture process. Then, a uniform cell population is obtained throughsubcloning using a limited dilution technique, after which hybridomacapable of producing an antibody specific to the peptide of the presentinvention may be mass-cultured in vitro or in vivo using a standardtechnique. The phage antibody library process may be performed in amanner in which an antibody gene for the peptide of the presentinvention is obtained and expressed in the form of a fusion protein onthe surface of a phage to thus manufacture an antibody library in vitro,after which a monoclonal antibody that binds to the peptide of thepresent invention is separated from the library and thus produced. Theantibody thus produced may be separated through centrifugation,electrophoresis, dialysis, ion exchange chromatography, affinitychromatography, and the like.

The antibody may comprise a functional fragment of an antibody moleculeas well as a complete form having two full-length light chains and twofull-length heavy chains. A functional fragment of an antibody moleculeis a fragment having at least an antigen-binding function, and includesFab, F(ab′), F(ab′)2, F(ab)2, Fv and the like.

In addition, the present invention pertains to a method of preparing theantibody, which is described below.

Preferably, the method includes (step 1) preparing an antigen forantibody production by linking the peptide of the present invention witha carrier protein using a crosslinker;

(step 2) mixing and emulsifying the antigen with an adjuvant;

(step 3) intradermally injecting the antigen containing the emulsifiedadjuvant to an animal two to five times at an interval of 7 to 20 days;

(step 4) separating the serum from the whole blood of the animal 7 to 20days after antigen administration; and

(step 5) separating and purifying an immunoglobulin from the serum.

In the method of preparing the antibody as above, the animal may be anyanimal capable of causing an immune response, and is preferably amammal. The immunoglobulin in step 5 may be of any type, but ispreferably immunoglobulin G.

When the immune response is induced using peptide 1, 2 or 3 in order toprepare the antibody of the present invention, the peptide 1, 2 or 3 islinked with the carrier protein using the crosslinker and may thus beused as an antigen. Here, the carrier protein may include BSA, KLH, OVA,etc., having low antigenicity and being responsible only for a carryingfunction, and the crosslinker for connecting the peptide and the proteinmay include EDC, glutaraldehyde (linkage of carrier molecules toN-terminus of peptide), succinimide esters (e.g. MBS, SMCC), benzidine(BDB) (linkage to Tyr residues), periodate (attachment to carbohydrategroups), isothiocyanate (used to label antibodies with fluorochromes),etc. As such, a buffer solution may be used in a variety of mannersdepending on the characteristics of the linker that is used.

In addition, the present invention pertains to a method of diagnosingprostate cancer using the peptide of the present invention or theantibody specifically recognizing the same.

The diagnosis method includes:

(step 1) separating a body protein from a sample to be analyzed;

(step 2) forming an antigen-antibody complex by bringing the bodyprotein of step 1 into contact with the antibody of the presentinvention; and

(step 3) quantitatively detecting and analyzing the antigen-antibodycomplex formed in step 2.

In the diagnosis method, the sample of step 1 may be extracted from asubject for whom the occurrence or progression of prostate cancer is tobe confirmed, and preferable examples thereof include tissue, cells,etc. of a mammal (human or other animals), and more preferable examplesthereof include urine, blood, plasma, serum, and liver cells.

Separating the body protein in step 1 may be performed using any knownprocess, and the amount of the body protein may be measured through anyof various methods known to those skilled in the art.

The antigen-antibody complex of step 2 is configured such that the EN2protein contained in the body protein of the sample (or Peptide 1, 2 or3 of the present invention) and the antibody are specifically bound toeach other. In the complex, the antigen means the EN2 protein (orPeptide 1, 2 or 3 of the present invention).

Through the above analysis method, the amount of the antigen-antibodycomplex formed in the case of a control and the amount of theantigen-antibody complex formed in the case of the subject for whom theoccurrence or progression of prostate cancer is to be confirmed may becompared, and the expression level of the EN2 protein of the subject forwhom the occurrence or progression of prostate cancer is to be confirmedis determined, and thus prostate cancer may be directly diagnosed.

In step 3, the amount of EN2 of the sample of the subject for whom theoccurrence or progression of prostate cancer is to be confirmed may bedetermined based on the standard value in which the concentration of therecombinant EN2 protein is measured. Here, detection of EN2 protein invivo using all of three antibodies, rather than one or two antibodies,is most preferable. In detail, protein taken from the sample may bedivided into three sets, and respective antibodies may react withproteins corresponding to respective sets, whereby EN2 may be detected.

The expression level of the EN2 protein of the subject for whom theoccurrence or progression of prostate cancer is to be confirmed may bedetermined by measuring whether it falls in the range of 3.1 to 65.4 nM(Sci. Rep. 2013; 3:2059), which is the known concentration range of EN2protein in the urine of prostate cancer patients.

The amount of the antigen-antibody complex that is formed may bequantitatively measured based on the signal magnitude of a detectionlabel. The detection label may be selected from the group consisting ofan enzyme, a fluorescent substance, a ligand, a luminescent substance,microparticles, a redox molecule and a radioisotope, but the presentinvention is not limited thereto. When an enzyme is used as thedetection label, examples of the enzyme include, but are not limited to,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase,alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinaseand the like. Examples of the fluorescent substance include, but are notlimited to, fluorescein, phycocyanin, fluorescamine and the like.Examples of the ligand include, but are not limited to, biotinderivatives, etc. Examples of the luminescent substance include, but arenot limited to, luciferin and the like. Examples of the microparticlesinclude, but are not limited to, colloids, gold and the like. Examplesof the redox molecule include, but are not limited to, quinone,1,4-benzoquinone, hydroquinone and the like. Examples of theradioisotope include, but are not limited to, ³H, ¹⁴C and the like.

Examples of the diagnosis method may include, but are not limited to,western blot, ELISA (enzyme linked immunosorbent assay), RIA(radioimmunoassay), radioimmunodiffusion, ouchterlony immunodiffusion,rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation,complement fixation assay, FACS (fluorescence-activated cell sorter),protein chip, etc.

As a subject to be diagnosed, to which the peptide or antibody of thepresent invention may be applied, any subject may be used so long as anEN2 protein may be produced in vivo, and diagnosis is possible for allmammals, including humans. The mammal may be any type of mammal, such asa human, a dog, a cat, a rabbit, cattle, a goat, a pig, a horse, etc.

Advantageous Effects

The present invention pertains to an immunogenic fragment peptide of anEN2 protein or an antibody composition specifically recognizing thesame. In the present invention, quantification of the EN2 proteinbecomes possible through a method of specifically recognizing thepeptide. In addition, the antibody prepared using the peptide is veryhigh in detection sensitivity compared to existing EN2 proteinantibodies, and can thus be efficiently used in a diagnostic agent fordiagnosing prostate cancer. In particular, when prostate cancer isdiagnosed, high diagnostic efficacy can result compared to results ofPSA (prostate-specific antigen) detection in the blood, which is aconventional prostate cancer diagnosis method.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows, in the entire peptide sequence of homeoboxprotein engrailed-2 (EN2), three types of peptide sequences used asantigens and antigen peptide sequences for commercially availableantibodies (the peptide of SEQ ID NO: 1: Peptide 1; the peptide of SEQID NO: 2: Peptide 2; the peptide of SEQ ID NO: 3: Peptide 3; the peptideof SEQ ID NO: 4: Antigen for preparing Company A antibody; and thepeptide of SEQ ID NO: 5: Antigen for preparing Company B antibody);

FIG. 2 schematically shows the overall timetable for an immunized animalin which an immune response is induced;

FIG. 3 (a to c) shows the results of SDS-PAGE of samples separated andpurified from the immunized animal serum;

FIG. 4 (a and b) shows the results of western blotting assay of theselective detection for EN2 protein expressed in a prostate cancer cellline (PC3) (in FIG. 4, the antibody for the peptide of SEQ ID NO: 1 isrepresented as Peptide 1, Pep.1, the antibody for the peptide of SEQ IDNO: 2 is represented as Peptide 2, Pep.2, the antibody for the peptideof SEQ ID NO: 3 is represented as Peptide 3, Pep.3, the antibody for thepeptide of SEQ ID NO: 4 is represented as Company A, ComA, and theantibody for the peptide of SEQ ID NO: 5 is represented as Company B,ComB);

FIG. 5 (a and b) shows the results of western blotting assay of theselective detection for EN2 protein expressed in a prostate cancer cellline (LNCaP);

FIG. 6 (a and b) shows the quantitative results of western blottingassay of the antigen-detectable concentration by each antibody usingrecombinant EN2 protein;

FIG. 7 (a and b) shows the results of western blotting assay of theantigen-detectable concentration in the urine using recombinant EN2protein;

FIG. 8 (a to c) shows the results of immunocytochemistry testing of thedetection of the purified antibody using recombinant EN2 protein;

FIG. 9 (a to c) shows the results of ELISA of the dissociation constantfor the binding capacity of the purified antibody and the antigen usingrecombinant EN2 protein;

FIG. 10 shows the results of ELISA measurement of affinity of thepurified antibody for the antigen using recombinant EN2 protein;

FIG. 11 schematically shows a process of evaluating the presence andexpression level of the EN2 protein, which is a biomarker for thediagnosis of prostate cancer in the urine, using an antibody compositionof the present invention;

FIG. 12 shows the quantitative analysis results of PSA(prostate-specific antigen) through ELISA from the blood in threeprostate cancer patients; and

FIG. 13 shows the results of western blotting assay of the presence andexpression level of the EN2 protein, which is a biomarker for thediagnosis of prostate cancer in the urine, using the antibodycomposition of the present invention.

MODE FOR INVENTION

A better understanding of preferred embodiments of the present inventionwill be given through the following examples. However, the presentinvention is not limited to these examples but may be embodied in otherforms. These examples are provided to thoroughly explain the inventionand to sufficiently transfer the spirit of the present invention tothose skilled in the art.

Example 1. EN2 Peptide Selection, Synthesis and Antigen PreparationExample 1-1. Selection of Amino Acid Sequence of Peptide Serving asAntigen and Synthesis of Peptide

Using a professional antibody company program (Antigen profiler peptidetool—Thermo Fisher Scientific), the entire protein sequence of EN2 wascut into peptides 30 amino acids long, and each peptide was scored forantigenicity ranging from 1 to 5 based on an antigenic index, amongwhich regions having a score of 3.8 or higher were selected. Here, theantigenic index is a measure of the probability that nucleotides areantigenic depending on the structural effect, the stereostructure andthe like caused by modification after translation thereof into proteins.

Among the regions thus selected, regions in which many hydrophobicresidues were distributed were excluded from the selection becauseexternal exposure is blocked in the folded protein structure, and amongthe top 10 peptides that scored most highly in the sequences of thepeptides selected based on the above criteria, three peptides having asmall number of hydrophobic residues were selected.

The above three peptides were requested to be synthesized in 10 mg at98% purity (Anigene, Korea), and three peptides comprising the aminoacid sequences of SEQ ID NOS: 1 to 3 were obtained. The positions of thepeptide sequences thus synthesized in the EN2 protein are shown in FIG.1.

SEQ ID NO: 1:  PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS SEQ ID NO: 2: CTRYSDRPSSGPRSRKPKKKNPNKEDKRPR SEQ ID NO: 3: PRSRKPKKKNPNKEDKRPRTAFTAEQLQR

Example 1-2. Linkage of Carrier Protein and Peptide

The three peptides prepared in Example 1-1 have small sizes, and whenthese peptides are directly used as antigens, it is difficult to inducean immune response, and thus each peptide was linked with a carrierprotein (KLH (keyhole limpet hemocyanin)) using a crosslinker.

Particularly, each peptide, a carrier protein, and a crosslinker weredissolved at a weight ratio of 1:1:1 and allowed to sufficiently reactat room temperature for 2 hr or more. As such, the crosslinker was EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), and as abinding buffer, 0.1 M MES (2-[N-morpholino]ethane sulfonic acid) (pH 4.5to 5) was used (the buffer functions to induce hydrolysis of an aminegroup).

After termination of the reaction, the bound material and the unboundmaterial were separated from each other using a Thermo Scientific ZebaSpin Desalting Column (#89891) based on a size difference therebetween(KLH 400 kD and peptide 3.5 kD, based on the size difference between abound material and an unbound material).

Then, the peptide thus linked with the carrier protein was used as anantigen for antibody preparation.

Example 1-3. Preparation of Antigen Emulsified with Adjuvant

In order to administer an antigen to an animal to be immunized, anantigen having high solubility is required to be coupled with anadjuvant so as to reside for a long period of time in vivo, and thus acomposition in which an adjuvant and an antigen were emulsified wasprepared.

Also in order to induce an effective immune response, the antigenprepared in Example 1-2 was administered a total of four times, and theantigen for first administration was prepared by mixing a Freund'scomplete adjuvant (FCA) containing killed mycobacterium in order tomaximize an immune response, and the antigens for the remaining threeadministrations were prepared by mixing a Freund's incomplete adjuvant(FIA) excluding killed mycobacterium.

Here, in order to effectively emulsify the water-soluble antigen and thehydrophobic adjuvant, the antigen and the adjuvant were mixed throughprobe sonication, and the temperature was maintained at 4° C. so as notto apply heat to the antigen during the mixing. The antigen and theadjuvant were mixed at a volume ratio of 1:1.

Example 2. Induction of Immune Response Using Antigen Example 2-1.Selection of Animal to be Immunized

Rats (Wistar) were used as animals in order to induce an immune responseto a human EN2 protein sequence. An immune response was induced in nine7-week-old female individuals for each peptide sequence in considerationof individual specificity, because the immune response may varydepending on individuals.

Example 2-2. Experimental Timetable and Injection Method for ImmunizedAnimal

The overall timetable for immunity induction followed the procedure ofFIG. 2, in which the antigen was first administered and was administeredagain after 2 weeks, after which the antigen was further administeredtwo times at an interval of 10 days, whereby a total of fouradministrations was performed. The extent of immune response was checkedthrough tail vein bleeding after the second administration (testbleeding). Intradermal injection was used as the method ofadministration, and 2 to locations were selected at the time of a singleadministration, and a total of 200 μL was injected per individual.

Example 3. Separation of Serum from Immunized Animal and Purification ofImmunoglobulin G (IgG) Example 3-1. Separation of Serum from ImmunizedAnimal

The immunized animals administered with the antigen in Example 2-2 weresacrificed as shown in FIG. 2. Whole blood was obtained throughabdominal vena cava blood collection using an inhalation anesthetic(Isoflurane). The whole blood was maintained at 37° C. for 1 hr and thencentrifuged at 2000 rpm for 20 min, and 4 to 5 mL of serum perindividual was obtained from the centrifuged supernatant.

Example 3-2. Separation of Immunoglobulin G (IgG)

Since the serum contains various proteins including albumin,immunoglobulin G (IgG) was purified alone in order to increase the titerand specificity of the antibody.

Separation and purification were performed using a resin coupled withprotein G specifically binding to rat IgG (Protein G Sepharose 4 FastFlow—GE Healthcare). The separated serum was diluted with a buffer (PBS)at a volume ratio of 1:1, bound to the packed resin, and washed with abuffer in a volume corresponding to 2 to 3 times the volume of beads andthen eluted.

As an elution buffer, a buffer having a pH of 2 to 3 was used to breakthe specific binding of protein G and IgG. In order to raise the pH to anormal level immediately after elution, Tris (pH 9) was used.

The serum was eluted into 10 fractions, and as is apparent from theSDS-PAGE gel, IgG was obtained from 7 to 9 fractions (FIGS. 3a and 3bshow the results of Peptide 3, and specifically show that the serumsample identified through the serum albumin was fractionated in FIG. 3aand thus the presence of IgG was confirmed in FIG. 3b ). For eachpeptide, as shown in FIG. 3c , final IgG was found to have high purityby removing other proteins from the serum per peptide (Whole: wholeantibody, HC: heavy chain of antibody, LC: light chain of antibody). Theantibody (IgG) of each of the finally obtained peptides was quantified,diluted with a PBS/0.2% sodium azide/20% glycerol buffer, aliquoted andstored at −80° C. Here, the amount of IgG obtained for respectivepeptide sequences and individuals is shown in Table 1 below.

TABLE 1 Ag Mouse Conc. (mg/mL) Volume (μL) EN2-1 1 10 500 2 10 300 3 10500 4 10 500 5 3 500 6 10 500 7 10 500 8 10 500 9 10 500 EN2-2 1 10 5002 10 300 3 8 300 4 10 500 5 10 500 6 10 500 7 10 300 8 10 500 9 10 300EN2-3 1 10 500 2 10 300 3 10 500 4 10 500 5 5 500 6 5 300 7 10 500 8 7300 9 10 500

Example 4. Expression and Purification of Recombinant Human EN2 Protein

In order to test the sensitivity and accuracy of the prepared antibody,recombinant EN2 protein was prepared. Particularly, pET28b/EN2 plasmidwas transformed into Escherichia coli (BL21/DE3) and thus EN2 proteinwas overexpressed under conditions of 0.1 mM IPTG and 37° C. Theoverexpressed Escherichia coli cells were lysed through sonication witha lysis buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 20 mM imidazole, a1× protease inhibitor cocktail, 1 mg/mL lysozyme) and centrifuged, thusobtaining only a water-soluble protein. The water-soluble protein wasaffinity-bound to EN2/His Tag and Ni on Ni-NTA agarose beads.Thereafter, EN2 protein was isolated with an elution buffer (20 mMTris-Cl at pH 8.0, 300 mM NaCl, 300 mM imidazole, a 1× proteaseinhibitor cocktail), and the imidazole was removed through dialysis(cutoff 10 K) in a storage buffer (50 mM Tris-HCl at pH 8.0, 200 mM KCl,0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol), followed by BCA(bicinchoninic acid) quantification and protein concentrationquantification at an absorbance of 280 nm.

Experimental Example 1. Evaluation of EN2 Protein Detection Ability inProstate Cancer Cell Line (PC3 Cell Line)

A prostate cancer cell line PC3 (®CRL-1435™) for use in experiments waspurchased from ATCC (American Type Culture Collection, Rockville, Md.,USA) and cultured in a 5% CO₂ humidified incubator at 37° C. with a 25mM HEPES-containing RPMI-1640 (PC3) or RPMI-1640 (LNCaP) medium addedwith 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

The antigen detection ability of the antibody (obtained in Example 3) ina prostate cancer cell line PC3 having high EN2 expression was measuredthrough western blotting assay. Here, the antibodies prepared in thepresent invention and commercially available EN2 antibodies (Company A,Company B) were compared and tested.

Company A: Thermo Fisher Scientific (PA5-14363)

Company B: Novus Biologicals (H00002020-M03)

Particularly, the cultured PC3 was lysed in a RIRA buffer containing aprotease inhibitor cocktail and centrifuged at 12000 rpm for 20 min tocollect the water-soluble protein, and the total protein content wasdetermined through a BCA quantification method.

More particularly, 2×10⁶ PC3 cells were cultured in 100 cm² culturedishes for 3 days and then washed two times with a cold PBS (phosphatebuffered saline) solution to thus collect the cells, which were thenlysed in ice for min in a RIPA buffer solution containing a proteaseinhibitor cocktail. The cell lysate was centrifuged at 13,000 rpm for 20min, and the protein concentration in the supernatant was measuredthrough a BCA method, and the lysed proteins were separated using 4-15%SDS-PAGE. The proteins separated through PAGE were transferred to a PVDFmembrane, and each EN2 antibody was diluted with 5% skim milk/TBS-T(Tris-saline+Tween20) at a ratio of 1:2000 and reacted overnight at 4°C., and the next day washing was performed three times with TBS-T, and ahorseradish peroxidase (HRP)-conjugated anti-RAT antibody was dilutedwith 5% skim milk/TBS-T at a ratio of 1:2000 and allowed to react atroom temperature for 2 hr. The membrane washed three times with TBS-Twas treated using an ECL (enhanced chemiluminescence) solution, and thusblue light emitted while luminol, which is a substrate of the peroxidasebinding to the secondary antibody, was oxidized by the peroxidase wasphotosensitized to an X-ray film.

As such, 20 μL of whole protein extracted from the PC3 cells in anSDS-PAGE gel was loaded at 40 μg/well, 20 μg/well, and 10 μg/well ineach well of a 96-well plate, followed by western blotting assay, afterwhich the protein detection ability was measured using the antibodies(6.6 nM) having the same concentration. The results are shown in FIG. 4.FIG. 4a shows the band images obtained through western blotting, andFIG. 4b is a graph showing the numeric values of the results.

As shown in FIG. 4, the antibodies of the present invention (prepared inExample 3 using Peptides 1 to 3 as antigens) exhibited a very highability to detect EN2 protein compared to commercially availableantibodies (Company A, Company B) (under protein treatment conditions of10 μg/well, the antibodies prepared using Peptides 1 to 3 as antigensexhibited detection ability 2 to 10 times as high as commerciallyavailable antibodies).

Experimental Example 2. Evaluation of EN2 Protein Detection Ability inProstate Cancer Cell Line (LNCap Cell Line)

As another prostate cancer cell line known to have high EN2 expression,LNCaP (®CRL-1740™) cells were used, and the detection ability of theantibodies was evaluated under the same conditions as in ExperimentalExample 1. The results are shown in FIG. 5. FIG. 5a shows the bandimages obtained through western blotting, and FIG. 5b is a graph showingthe numeric values of the results.

As shown in FIG. 5, compared to commercially available antibodies(Company A and Company B), the antibodies of the present invention(against Peptides 1 to 3 prepared in Example 3) exhibited superiorselective antigen detection ability. Thus, based on the results ofExperimental Examples 1 and 2, the antibodies prepared in Example 3showed selective binding to the EN2 antigen, rather than cell-specificbinding.

Experimental Example 3. Evaluation of Sensitivity Using Recombinant EN2Protein

In lieu of the prostate cancer cell protein, the human EN2 recombinantprotein prepared in Example 4 was diluted at concentrations from amaximum of 50 ng/20 μL (2.5 ng/μL) (60 nM) to a minimum of 5 ng/20 μL(0.25 ng/μL) (6 nM) and the sensitivity of the antibodies was measuredunder the same conditions as in Experimental Example 1. The results areshown in FIG. 6. FIG. 6a shows the band images obtained through westernblotting, and FIG. 6b is a graph showing the numeric values of theresults.

As shown in FIG. 6, all of the antibodies for three types of peptidesexhibited EN2 protein detection sensitivity to a minimum of 5 ng/20 μL(0.25 ng/μL) (6 nM). This indicates that the antibodies prepared in thepresent invention have antibody sensitivity able to detect the EN2protein concentration in actual patient urine (EN2 protein concentrationrange in the prostate cancer patient urine: 3.1 to 65.4 nM: Sci. Rep.2013; 3: 2059).

Experimental Example 4. Evaluation of EN2 Protein Sensitivity in Urine

Considering that the sample from the subject to be diagnosed in thepresent invention is the urine of a prostate cancer patient, whether itwas possible to detect EN2 protein in the secreted urine was tested.Particularly, recombinant EN2 protein was added to urine from a normalperson at a concentration determined to be typical for urine of aprostate cancer patient, and was used immediately as a sample.

Here, the concentration of the recombinant protein and the entireexperimental procedure were the same as in Experimental Example 3. Theresults are shown in FIG. 7. FIG. 7a shows the band images obtainedthrough western blotting, and FIG. 7b is a graph showing the numericvalues of the results.

As shown in FIG. 7, despite the effects of organic/inorganic materialscontained in the urine, all of the respective antibodies recognizing thethree peptides were able to detect the antigen up to a minimumconcentration of 0.25 ng/μL (6 nM).

Experimental Example 5. Evaluation of Detection Ability ThroughImmunofluorescence Staining Testing in Cell Line (LNCap)

The intracellular EN2 protein of the prostate cancer cell line (LNCap)was detected based on antigen-antibody specificity. As the primaryantibody, the three types of antibodies prepared in Example 3 were used,and as the secondary antibody, anti-rat IgG/FITC was used, and thebinding capacity to the antigen distributed in the cytoplasm wasanalyzed through confocal laser microscopy. The fluorescence stainingimages thereof are shown in FIG. 8. FIG. 8a shows the results of Peptide1, FIG. 8b shows the results of Peptide 2, and FIG. 8c shows the resultsof Peptide 3.

As shown in FIG. 8, all of the three types of antibodies can beconfirmed to bind to the EN2 protein distributed in the cells, wherebythe antibody of the present invention can be easily used, even forantigen detection under non-denaturation conditions.

Experimental Example 6. Evaluation of Sensitivity Using Recombinant EN2Protein (K_(d): Dissociation Constant)

The recombinant EN2 protein was attached to a 96-well plate at 4° C.overnight, and blocking was carried out at 37° C. using a TBST solutioncontaining 2% skim milk. Here, the experiment was progressed under thecondition that the recombinant EN2 protein was fixed at 250 ng/well andthe antibody was subjected to serial dilution (FIG. 9), and theexperiment was progressed under the condition that the antibody wasfixed and the antigen was diluted at 0.5 to 50 ng/mL (FIG. 10).

Thereafter, each well was treated with the primary antibody, and with,as the secondary antibody, HRP-conjugated anti-rat IgG diluted at1/10000. After the reaction, a color development reaction was progressedusing a TMB (3,3′,5,5′-tetramethylbenzidine) solution and terminatedusing a 1 N sulfuric acid.

The resultant values were quantified using an ELISA reader and the Kdvalue was calculated using the association kinetics method of the Prismprogram. The results are shown in FIG. 9, and the EN2 content detectedby each antibody is shown in FIG. 10.

As shown in FIG. 9, the dissociation constant Kd is the amount ofantigen required to occupy ½ of the antibody-binding site, and anantibody having high affinity for an antigen has a small dissociationconstant. The Kd values of the antibodies are as follows: Peptide 1:4.087×10⁻¹³, Peptide 2: 5.825×10⁻¹², and Peptide 3: 7.739×10⁻¹³. Also,the antibody for Peptide 3 showed the highest value, and compared to anormal natural antibody having a Kd value of 10⁻⁷ to 10⁻¹⁰, thedeveloped antibody had a Kd value of 10⁻¹² to 10⁻¹³ and thus manifesteda high titer, whereby the antibody (prepared in Example 3 according tothe present invention) having high binding capacity to the recombinantEN2 protein antigen was confirmed to be prepared.

With reference to FIG. 10 and Table 2 below, all of three antibodieswere able to detect EN2 antigen up to a minimum concentration of 0.5ng/mL through ELISA.

TABLE 2 ELISA O.D. value (EN2 protein) 0 ng/mL 0.5 ng/mL 2.5 ng/mL 5ng/mL 10 ng/mL 25 ng/mL 50 ng/mL Pep1 0.22250 0.25425 0.28450 0.324250.42175 0.59700 0.86575 Pep2 0.22575 0.25775 0.32350 0.41600 0.629001.04650 1.68600 Pep3 0.21350 0.24575 0.28825 0.33400 0.46075 0.742751.09133

Therefore, these results show that the antibody of the present inventionis deemed to be an excellent antibody because it can be utilized notonly in a qualitative test to determine the presence of cancer, but alsoa quantitative test to evaluate the progression of cancer by quantifyingthe correlation between EN2 detection in the urine and cancerprogression.

Experimental Example 7. Evaluation of Efficacy in Prostate CancerPatient Urine Sample

In order to evaluate EN-2 detection ability in the prostate patienturine sample for use in actual tests, three prostate cancer patientsamples (P1, P2, P3) were centrifuged at 10000 g for 10 min and thesupernatant was isolated and subjected to western blotting. 15 μL of theurine sample was mixed with 5 μL of a 4× sample buffer and boiled at100° C. for 5 min, and western blotting was carried out. Here, theconcentration of the antibody used was 3.3 nM, and as a negative control(H.C.), urine from healthy males was used. The results are shown in FIG.13. The PSA expression results of prostate cancer patients, obtainedthrough ELISA as in Experimental Example 6, are shown in FIG. 12.

With reference to FIG. 13, all of the three types of antibodies of thepresent invention can be concluded to exhibit high EN-2 detectionability in actual patient samples.

The invention claimed is:
 1. A polyclonal antibody compositionspecifically recognizing a peptide consisting of the amino acid sequenceof SEQ ID NO: 1 (PGDGEGGSKTLSLHGGAKKGGDPGGPLDGS), wherein the peptide isan immunogenic fragment of an EN2 (engrailed-2) protein.
 2. A diagnosticagent for diagnosing prostate cancer, containing the antibodycomposition of claim
 1. 3. A method of diagnosing prostate cancer, usingthe antibody composition of claim 1.